Protein glycosylation is mediated by a series of enzymes found in the Golgi apparatus. Many of the enzymes in this pathway are subject to regulation during embryogenesis, lymphocyte activation, and in cancer progression. Structural diversity of carbohydrates on cell surfaces and secreted or non-secreted (e.g. receptors) proteins affects their function and the associated cell biology. Somatic mutations and drugs which block the biosynthesis of -GlcNAcβ1-6Manβ-branching of N-linked oligosaccharides, also inhibit organ colonisation, invasion in vitro, and limit solid tumor growth in vivo.
Synthesis of GlcNAc-branched carbohydrate structure is dependent upon N-acetylglycosaminyltransferases, one of which is N-acetylglycosaminyltransferase V (GlcNAc-TV). GlcNAc-TV catalyzes the addition of 1-6GlcNAc to thetrimannosyl core in the biosynthetic pathway for branched complex-type N-linked oligosaccharides found on some cell surface and secreted glycoproteins (Schachter, H. (1986) Biochem. Cell. Biol. 64: 163-181). The 1-6GlcNAc product of GlcNAc-TV is the preferred antenna and rate limiting substrate in the pathway for addition of terminal polylactosamine sequences which affect cell-cell and cell-substratum interactions (van den Eijnden, D. H. et al, (1988) 263:12461-12465; Yousefi, S. et al, (1991) J. Biol Chem. 266:1772-1783; and Heffernan, M. et al, (1993) J. Biol. Chem. 268:1242-1251).
The rat (Shoreibah, M. et al (1993), 268:15381-15385) and human (Saito, H. et al. (1994), Biochem. Biophys. Res. Commun. 198:318-327 233:18-26) GlcNAc-TV sequences predict a 741 amino acid type II glycoprotein. The human GlcNAc-TV gene is located on human chromosome 2q21 with 17 exons and spans 155Kb (Saito et al., (1995) Eur. J. Biochem. 233: 18-26). The putative promoter region of the GlcNAc-TV gene has AP1 and PEA3/ets binding sites, and is responsive to ras signaling pathways (Buckhaults P J Biol Chem (1997) 272:19575-19581).
Oncogenic transformation of rodent fibroblasts by polyoma virus, v-src, H-ras or v-fps leads to increased GlcNAc-TV expression (Yamashita, K. et al, (1985) J. Biol. Chem. 260:3963-3969; Pierce, M and Arango, J. (1986) J. Biol. Chem. 261: 10772-10777, Dennis, J et al. (1987) Science 236::582-585, 1987), and in human carcinomas of breast, colon and skin GlcNAcTV-generated structures correlate with pathological staging of tumors (Fernandes, B. et al (1991) 51:718-723). The GlcNAc-TV message is also subject to increased frequency of alternate splicing in tumors cells, resulting in a peptide encoded by an intron sequence of the GlcNAc-TV gene which has been identified as a widely occurring “tumor-associated antigen”. Fifty percent of tested human melanoma tumors expressed this antigen, while it is absent in normal tissues (Guilloux, Y. et al (1996) J. Exp. Med. 183:1173-1183). In a rat model of heritable liver cancer, GlcNAc-TV transcript levels are elevated in primary tumors and lymph node metastases (Miyoshi, e. et al, (1993) Cancer Res. 53:3899-3902, 1993). In addition, topical expression of GlcNAc-TV in epithelial cells results in morphological transformation and tumorogenesis (Demetriou, M. et al (1995) J. Cell Biol. 130:383), while tumor cell mutants selected for loss of GlcNAc-TV activity show reduced malignant potential in vivo (Lu, Y. et al (1994) Clin. Exp. Metastasis 12:47-54).